Despite the fact that the nonsteroidal estrogen diethylstilbestrol (DES) exerts its toxic effects primarily on the reproductive system, little is known about the possible interference of this compound with germ cell DNA. The measurement of unscheduled DNA synthesis (UDS) in spermatocytes and early spermatids of mice germ cells is a valid indicator for the DNA-damaging potential of a compound. UDS occurrence was thus determined after IP administration of 10, 30, 60 or 180 mg/kg DES to male mice. Tritiated thymidine ( [3H]dThd) was then injected into the testes, the spermatozoa were serially collected, the sperm heads isolated, and UDS determined by the amount of [3H]dThd incorporation. [3H]dThd measurements in germ cells of mice which were treated with 10 mg/kg DES were comparable to those of the controls. Higher incorporation of [3H]dThd, indicating UDS, was measured in sperm cells which had been spermatocytes at the time of treatment with 30 and 60 mg/kg DES; this increase was statistically significant at 60 mg/kg. Administration of 180 mg/kg DES caused [3H]dThd incorporation which was comparable to that of the controls, suggesting that DES interfered with repair mechanisms or delayed spermatogenic cycles at high dose levels. General toxicity was manifested in a dose-dependent decrease of the sperm cell numbers in the spermatogenic stages investigated. This study provides evidence that DES, or its metabolite(s), reached the germ cells of adult mice in sufficient amounts to produce DNA damage. The levels of radioactivity measured were comparable to those measured after cyclophosphamide treatment, but [3H]dThd incorporation was about 10 times less than in methylmethane sulfonate-treated animals.